Search

We purified CA125 antigen from the conditioned media (CM) of eutopic and heterotopic endometrial epithelial cells (EC) as well as ovarian cancer cell lines, SHIN-3 and HOC-I, to determine what molecular weight forms of CA125 antigen were identifiable. Treatment of the high-molecular-weight CA125 antigen with 6M urea yielded a much lower molecular mass peak. After purification by OC125 affinity column chromatography, samples were applied to 3 to 7% polyacrylamide gradient gel and analyzed by Western blot. A single band with a molecular weight (MW) of 200KDa was identified in eutopic EC materials. The CA125 polypeptide of the 110KDa molecule could be detected in all of the CM obtained from heterotopic EC, irrespective of the length of time in the cell culture. A MW of approximately 200 KDa was also observed in some heterotopic EC samples. On the other hand, although the multiple bands with a MW equal to orless than 200KDa were observed in the CM of two ovarian cancer cells, the CA125 polypeptide of 110KDa molecules could not be detected. This preliminary finding offers promise that the 110KDa molecule detection method may be a useful adjunct in the differential diagnosis of heterotopic EC and ovarian cancer.

We have established an in vitro system in which epithelial cells (EC) and stromal cells (SC) from the human endometrium are placed in culture to examine the production of CA125. There was a much greater amount of CA125 in heterotopic EC after culture cells reached confluence than during the logarithmic growth phase. Heterotopic EC in culture secreted nine times as much CA125 constitutively after reaching confluence as eutopic EC. Flow cytometry (FCM) analysis indicated that the changes in CA125 expression correlated with the cell cycle. The CA125 expression was mainly observed in the G0/G1-phase in the cell cycle. There was no amplification of the CA125 expression in heterotopic EC membranes. The results of SDS-PAGE and Western blot indicated that the 110 KDa molecule of CA125 might be specific for adenomyosis. The biochemical and physical nature of CA125 was examined to characterize the antigenic determinant of this antigen. These results strongly suggested that the CA125 antigenic determinant from EC was composed of conformationally dependent peptides. We conclude that significantly increased secretion of CA125 from heterotopic EC could be attributed to the increase in serum CA125 in patients with adenomyosis.

Six tumor-associated antigens, cancer antigen 125 (CA125), tissue polypeptide antigen (TPA), ferritin (Fr), carcinoembryonic antigen (CEA), alpha fetoprotein (AFP), and sialyl Lex-i (SLX) were measured simultaneously for the early detection of ovarian cancer. To decrease the number of both false positive and false negative cases in the combination assay, statistical discrimination analysis employing the serum values for appropriate tumor markers has been studied with respect to ovarian cancer by the method of Mahalanobis' generalized distance. The new "ovarian cancer screening test" designed by us has been used in Shizuoka Prefecture since 1988, and 23,307 serum samples have been analyzed. One hundred twenty-seven of 165 ovarian cancer patients were suspected as having cancer by such clinical procedures as pelvic examination and/or ultrasonography, while in 150 patients cancer was detected by the statistical discrimination method. Thirty-one of 38 patients with ovarian cancer overlooked by the clinical procedures could be found by the statistical method. We conclude that clinical procedures and the statistical method can be complementary in detecting patients with this malignancy.

Circulating serum sialyl Tn (STN) antigen levels were measured in 89 patients with epithelial ovarian cancer, 157 benign disease, and in 126 healthy controls. Serum antigen levels were increased in 48.3% of patients with ovarian cancer. The false positive rate is significantly low (4.0% in healthy controls and 9.6% in benign disease). The levels of STN antigen were significantly higher in sera of patients with cancer than in those in benign and healthy controls (p less than 0.05). The rise in serum STN antigen levels correlated to the size of the primary tumors. Of the histological type, it is interesting to note the high sensitivity in mucinous-type ovarian cancer. Survival at 1, 2, 3, 4 and 5 years for patients with STN-negative (serum STN levels less than 50 U/ml) versus STN-positive (serum STN levels greater than or equal to 50 U/mol) was 96.2, 92.3, 86.5, 82.7, and 76.9% versus 59.5, 29.7, 18.9, 10.8, and 10.8%, respectively (p less than 0.05). The overall survival probability was worse in patients with STN-positive sera. Percent progression-free survival at 1, 2, 3, 4 and 5 years for patients with STN-negative versus STN-positive was 90.4, 86.5, 76.9, 59.6, and 51.9% versus 35.1, 16.2, 8.1, 8.1, and 5.4%, respectively (p less than 0.05). The overall progression-free period of survival was shorter in patients with STN-positive sera. Multivariate regression analysis revealed that positive STN, stage, PS and histologic grade were the four variables of most importance in predicting survival. These results indicate that a positive STN antigen level in sera is an independent predictor of poor prognosis in ovarian cancer.

Clinically we rather frequently treat infertile women with endometriosis, either external or internal. Nowadays the study of the histogenesis of endometriosis is urgently required by patients who suffer from sterility because of endometriotic foci. To evaluate the histogenesis of endometriosis, we first electron microscopically examined the adenomyotic glandular epithelium as a distinct candidate for endometriosis interna. Furthermore we studied the chocolate cyst of the ovary as representative of endometriosis externa which is very important from the standpoint of the infertility clinic. In this report we carefully compared the ultrastructure of the adenomyosis with the ovarian chocolate cyst, and endeavoured to explore the histogenesis of endometriosis through this ultrastructural comparison. As a result it was found that the nuclear membrane showed tremendous irregularity in the ovarian chocolate cyst compared with the smooth nuclear membrane in the adenomyotic glandular epithelium.

Anticancer chemotherapy with cisplatin (CDD) as the main drug (combined with adriamycin (ADM) and cyclophosphamide (CPM), PAC therapy) was performed on patients with ovarian cancer. Urinastatin (US) was concurrently administered to assess its effectiveness in preventing CDDP-induced nephrotoxicity. Twenty-two patients with gynecological malignant tumor were treated with PAC therapy, and of these, twelve concurrently received US. The ten who did not receive US served as the control. As a rule, one course of PAC therapy consisted of 50mg/m2 CDDP, 50mg/m2 ADM and 500mg/m2 CPM. Before the administration of CDDP, US 100,000 units was administered by I.V. drip infusion and after the administration, US 400,000 units was again administered by I.V. drip infusion at a speed of 100,000 to 200,000 units/hour. A total of approximately 3,500ml of fluids was administered I.V.. Each course of PAC therapy took 7 to 14 hours to complete. The control group underwent PAC therapy in a regimen not including US. As indexes of nephrotoxicity, serum levels of BUN, creatinine (Cr), and creatinine clearance (Ccr), and N-acetyl-beta-glucosaminidase (NAG), gamma-glutamyl transpeptidase (gamma-GTP), and arylamidase (AA) activity in the urine was determined before treatment and at days 1, 2, 3, 7, 14, and 21 after the initiation of PAC therapy. Changes in serum BUN, Cr, and Ccr levels after CDDP administration in the group with and the group without concurrent US were similar. Urinary gamma-GTP, AA, and NAG activity remained unchanged after CDDP administration in the group with concurrent US. In contrast, in the group without US, this urinary enzyme activity was transiently increased after CDDP administration.(ABSTRACT TRUNCATED AT 250 WORDS)

The efficacy of two methods of chemotherapy in the treatment of patients with advanced ovarian cancer was compared on the basis of survival curves; one consisted of remission induction therapy alone with a combination of cisplatin (CDDP), adriamycin (ADM) and cyclophosphamide (CPM) (induction PAC therapy), and the other consisted of induction PAC therapy and additional maintenance therapy with cyclic PAC (cyclic PAC therapy). The subjects of the study were patients with advanced ovarian cancer in stages III and IV. Sixty-eight patients received induction PAC therapy alone and seventeen patients received both induction and cyclic PAC therapy. Demographic factors such as age at initial presentation, the stage of cancer (III or IV), surgical procedure, histological classification, number of courses of induction PAC, response rate, site of residual tumor after surgery and induction PAC therapy, and reduction rate of CA125 were compared in the two groups. When analyzed by the chi 2 test, none of these factors was significantly different in the two groups. Patients in the induction PAC therapy group received a median total dosage of CDDP 360 mg, ADM 235mg, and CPM 2.246mg. Patients in the cyclic PAC therapy group received CDDP 592mg, ADM 490mg, and CPM 4.642mg. Thus, the dosage of anticancer agents administered to the latter group was about twice as great as that administered to the former group. According to the Kaplan-Meier method, survival rates for the induction PAC therapy group were 88.2% for one year, 50.0% for two years, 28.0% for three years, and 8.8% for five years. The median survival period was 23 months.(ABSTRACT TRUNCATED AT 250 WORDS)

The inactivation of oxytocin and angiotensin II by retroplacental serum and placental leucine aminopeptidase (P-LAP), which had been highly purified from retroplacental serum, was demonstrated using biological methods.

We measured the six tumor markers, CA125, TPA, Fr, CEA, AFP, and PPA simultaneously in serodiagnostic testing as a method for the early detection of ovarian cancer. To decrease the false negativity in the combination assay, statistical discriminating analysis was applied to serum values for tumor markers. "The ovarian cancer screening test" designed by us has been enforced in Shizuoka Prefecture since September, 1985. Twenty-one thousand nine hundred serum samples had been analysed by December, 1988. Eighty-four cases out of the 125 patients with ovarian cancer were suspected of being malignant pretherapeutically in a clinical diagnostic procedure which included echography. On the other hand, malignancy was justified before treatment in 109 patients in the combination assay and statistical analysis. One hundred patients were diagnosed as malignant in the combination assay and 9 cases by statistical analysis. However, we overlooked 4 patients (3 patients were found to be in stage I and one in stage III, respectively) even when both methods were used. Altogether 37 more patients with ovarian cancer could be found by the serodiagnostic screening method with discriminating analysis using tumor markers than by clinical findings such as image diagnosis and others. To conclude, the discriminating analysis was, therefore, verified as a useful technique for the diagnosis of ovarian cancer.

Hitherto, the selection of anticancer drug has been based on clinical experience. But to succeed for chemotherapy to succeed, effective drugs must be selected for individual patients with cancer. We have developed the following new in vitro micromethod. Method. In brief, tumor cells obtained by surgery were incubated with anticancer drugs for 48 hrs at 37 degrees C in 5% CO2 in a microplate. After incubation, the surviving cells were fixed with methanol and stained with crystal violet. Then the stained cells were solubilized with 1% lauryl sulfate and the absorbance of each well was measured at 540 nm with a multiscan spectrophotometer. In this method, cytotoxicity was quantitated by the absorbance. The method is simple, rapid (48 hrs) and reproducible, and it requires only a small amount of cells (5 X 10(3) approximately 1 X 10(4]. This method correlates well with sensitivity tests using isotopes. We have examined the chemosensitivity of 22 specimens from gynecologic malignancies by this method. The success rate is 77% and higher than with any other in vitro method. This method will be widely utilized in several fields of cancer treatment in the near future.

The retrospective analysis of 68 advanced ovarian cancer cases was performed mainly to see whether certain predictive factors were present which might distinguish patients with risk for recurrence or death from those with a good prognosis. Six factors, such as the mode of operation, the site of metastasis, the degree of peritonitis carcinomatosa, the rate of the decline in serum CA125. Performance Status, and pathological type were selected as factors influencing prognosis. We then tried to tally the prognosis estimation score using these factors. The correlation between this score and the survival period was noted in only patients with a survival time up to 3 years. The following formula was obtained: Y = 1.286X -3.429 [X: prognosis estimation score, Y: survival period (months)]. Cases were classified as follows according to the this score. When patients had a value equal to or less than 17.84.2% of patients died within 21 months, while if patients had a value greater than 17.34.7% of cases survived more than 36 months. On the other hand, 89.5% of cases with a survival period longer than 36 months had a prognosis estimation score greater than 18. However, there was little significant correlation between this score and the survival period in patients with survival period longer than 3 years. To conclude, although a low value for this score in advanced ovarian cancer, indicates a poorer prognosis, we cannot make an accurate prognosis for more than 3 years from this score.

We discussed the mechanism of the increase in CA125 among patients with benign gynecologic diseases, especially with endometriosis. The tissue CA125 concentrations of surface endometrium in patients with adenomyosis were as follows; the highest tissue concentration was observed at the early proliferative phase followed by the late secretory one and was lowest in the late proliferative one. The tissue CA125 concentration showed the significantly different characteristics in surface and ectopic endometrium. The increase in CA125 in the intraperitoneal fluids was observed among cases of early pregnancy, acute appendicitis and ovarian hyperstimulation syndrome (OHSS). Tissue samples from both the peritoneum obtained from patients with acute appendicitis and the cyst wall obtained from OHSS cases showed a high concentration of tissue CA125. From these findings, it was suggested that one of the causes of serum CA125 increase in patients with adenomyosis appeared to be the increase in the ectopic endometrial tissues in the myometrium and direct shedding from ectopic endometrial cells into peripheral circulation. On the other hand, in patients with chocolate cysts, the increase in serum CA125 was suppressed because it was secreted into the inside of the chocolate cyst. The production of CA125 may take place not only from ectopic endometrial cells of adenomyosis but also from the peritoneal tissues of patients with acute appendicitis.

The present study was performed to evaluate the mechanism of the protective effect of urinastatin (US) against cisplatin (CDDP) induced nephrotoxicity. We measured consecutively the change in the creatinine clearance (Ccr) level, urinary arylamidase (AA) activity, and urinary gamma-glutamyl transpeptidase (gamma-GTP) activity as the indices of nephrotoxicity. These drugs such as US (10,000 U/kg, 3h: group 1), gabexate mesilate (2mg/kg/h, 4h: group 2), and aprotinin (5,000 U/kg/h, 4h: group 3) as protease inhibitors, and dopamine HCl (0.3mg/kg/h, 4h: group 4) for the increase in renal blood flow were used with the hydration of 15 ml/kg/h during 4 hours of experiments on rabbits. The change in the Ccr value after the intravenous administration of CDDP indicated that the level in groups 3, 4 decreased significantly at one day after the administration of CDDP. On the other hand, there was little significant increase in urinary AA activity and/or urinary gamma-GTP activity in group 1, while a transitional increase was observed in groups 2, 3, and 4, among which the highest level was found in group 4. These results are attributed to the protective action of US against CDDP mainly in the inhibition of the lysosomal enzyme released by the destruction of lysosomes in the proximal tubule cells of the kidney.

We measured five tumor markers simultaneously for serodiagnostic testing as a method for the early detection of ovarian cancer. To decrease both false negativity and false positivity in the results of combination assay, statistical analysis including factor analysis and stepwise discriminating function was applied in this study. At least one of these tumor markers was detected as positive in 75.2% (76 of 101 patients) of sera from patients with ovarian cancer before treatment. Six hundred and ninety-three of 7,097 normal sera (9.8%) gave spuriously positive combination assay results. Falsely positive combination assay results were observed in 1,107 of 3,139 patients with benign disease, which could largely be attributed to the high CA125 values in patients with endometriosis. On the basis of factor analysis in order to decrease false positivity, CA125, TPA, and CA125/TPA were selected as the best parameters for distinguishing between ovarian cancer and benign conditions including those pelvic endometriosis showing positive results in combination assays. Subsequently, the function derived by factor analysis made possible the correct classification of 58 of 71 patients with ovarian cancer detected by combination assay, and 84.8% of pelvic endometriosis and benign ovarian tumor subjects were correctly classified into the non-cancer group. Next, in order to decrease false negativity, statistical analysis was also applied. Malignancy could be correctly diagnosed by this procedure in 14 of 25 patients whose tumor was undetectable by combination assay, whereas of subjects without cancer 14.5% were errorenously classified into the ovarian cancer group. In the search for the best method for accurately detecting ovarian cancer, we used image diagnosis (ultrasonography) in combination with serological diagnosis.(ABSTRACT TRUNCATED AT 250 WORDS)

A serological diagnosis of ovarian carcinoma was performed using a combination assay consisting of three tumor markers. Cancer Antigen 125 (CA125), Sialyl Lex-i (SLX), and CA72-4. The results were compared with those for the individual tumor markers. Furthermore, the diagnostic accuracy of the combination assay was compared with that of image diagnosis in patients with stage I ovarian carcinoma. 1. The combination assay was positive in 90.3% of the patients with ovarian carcinoma. Classified according to the clinical staging system, the positive rate increased progressively with each stage, 77.6% in stage I, 92.0% in stage II, 98.5% in stage III, and 100.0% in stage IV. According to histological types, the positive rates were 93.8% in serous cystadenocarcinoma, 87.0% in mucinous cystadenocarcinoma, 88.9% in endometrioid carcinoma, and 85.7% in clear cell carcinoma. On the other hand, 6.9% of healthy persons and 38.6% of patients with various benign diseases were found to be false positive in this diagnosis. The high false-positive rate in the latter group is thought to result from the high false-positive rate of 73.5% and 57.1% for adenomyosis and pelvic endometriosis, respectively. 2. The accuracy of the image diagnosis and combination assay was compared in 58 cases with stage I ovarian carcinoma. Both procedures were positive in 38 cases (65.5%). Two cases (3.4%) were positive in image diagnosis but negative in the combination assay. Seven cases (12.1%) were negative in image diagnosis but positive in the combination assay.(ABSTRACT TRUNCATED AT 250 WORDS)

The patients with endometriosis have shown a significant increase in frequency in our country. In the recent years, the incidence of endometriosis in gynecological outpatients is probably 3%. In infertile patients, endometriosis has been demonstrated by laparoscopy in 20-30% of cases. The pregnancy rate of women with endometriosis has been noted to be 80-85% in the internal endometriosis and 50-60% in the external endometriosis. The risk of infertility is greater in women with external endometriosis than in women with internal endometriosis. The causes of infertility are multiple and additive. Mechanical interference with the reproductive function plays a predominant role. Pelvic adhesions involving ovaries and fallopian tubes interfere mechanically with ovulation, ovum pick-up and ovum transport. According to Brosens et al. the frequency of LUF syndrome is in excess of 79% in the patients with endometriosis. Another antifertility effect of endometriosis may involve coital avoidance due to dyspareunia, hyperprolactinemia, increased phagocytosis of spermatozoa and altered prostaglandin secretion in peritoneal fluid. Conservative treatment of endometriosis is associated with improvement infertility and the post-treatment pregnancy rate has become an acceptable indicator of the effectiveness of therapy. The choice of therapeutic approach depends on several factors including age of the patient, extent of the disease, extent and location of pelvic adhesions and other coexisting infertility factors. Several hormonal regimens have been recommended for the treatment of endometriosis. They include androgen, progestogens and pseudopregnancy, danazol-induced pseudomenopause, gestrinone and LH-RH agonists.(ABSTRACT TRUNCATED AT 250 WORDS)

The remission rate of choriocarcinoma has greatly improved since the introduction of effective multiagent chemotherapy combined with aggressive surgical therapy and radiotherapy. In addition to these, the registration and follow-up of gestational trophoblastic disease (GTD) also has been playing an important role in the early detection and treatment of choriocarcinoma following hydatidiform mole. The system for the registration and follow-up of GTD was started in 1977 in Shizuoka Prefecture. In the present series, the results obtained with this registration and follow-up system from 1977 to 1988 in Shizuoka Prefecture were reviewed and analysed. 1) One thousand, nine hundred and twenty-five cases of hydatidiform mole, 68 cases of invasive mole, 70 cases of persistent trophoblastic disease and 48 cases of choriocarcinoma were registered in 12 years. The overall registration rate was 97.4%. 2) The number of cases of hydatidiform mole registered has decreased from about 180 cases to about 140 cases per year, probably due to the decreasing birth rate. 3) The number of cases of choriocarcinoma registered has recently been decreasing significantly and the number of cases of registered invasive mole and persistent trophoblastic disease has decreased slightly. 4) Antecedent pregnancy with choriocarcinoma including clinical choriocarcinoma has been changing from "post-molar" to "post-term" in the past 12 years. The prognosis of the patient with choriocarcinoma following hydatidiform mole has improved by the early detection and treatment since the introduction of the registration and follow-up system. More attention should be paid to choriocarcinoma following term gestation not yet included in the registration and follow-up system to facilitate early detection and treatment.

The usefulness of tumor markers in serodiagnosis of cancer designed to detect ovarian cancer at an early stage was evaluated from the point of view of their diagnostic value. Namely, eight tumor markers, CA125, SLX, CA72-4, TPA, Fr, CEA, CA19-9, and SCC, were determined and studied to find the combination that would yield the optimal diagnostic value. For this purpose, the diagnostic value was calculated from sensitivity x specificity. As a single tumor marker CA125 proved optimal with a diagnostic value of 0.50. The higher value, 0.53, was obtained as the diagnostic value from the combination of two tumor markers, CA125 and CA72-4. When three tumor markers were combined, CA125, CA72-4 and SLX gave an optimal diagnostic value of 0.65. In the combination of four tumor markers, CA125, CA72-4, SLX and CA19-9 gave a diagnostic value of 0.63. In the five marker combination CA125, CA72-4, SLX, CA19-9 and TPA worked well and had a diagnostic value of 0.59. When the markers were increased to six types, CA125, CA72-4, SLX, CA19-9, TPA and Fr provided a combination with 0.53 as the diagnostic value. In the seven marker combination CA125, CA72-4, SLX, CA19-9, TPA, Fr and CEA gave a diagnostic value of 0.51. The efficiency declined to 0.51 when eight tumor markers were combined. When cost performance in the measurement of tumor markers for early detection of ovarian cancer is taken into account, a dilemma arises in that the increase in the number of tumor markers used is accompanied by higher sensitivity and lower specificity.(ABSTRACT TRUNCATED AT 250 WORDS)

16 cases with gestational choriocarcinoma treated at our clinic from 1978 to 1987 were reviewed in connection with the surgical treatment for metastases in the brain and lung. 9 patients were classified as high risk, 5 patients as medium risk and 2 patients as low risk according to Bagshawe's prognostic score. An overall remission rate of 87.5% was achieved. 10 patients underwent surgical treatment with chemotherapy in 14 of 16 patients in whom metastases were identified, followed by complete remission. Pulmonary metastases were noted in 14 of 16 patients and lobectomy was performed in 10 of 14 patients, resulting in complete remission. Brain metastasis also was noted in 3 of 16 patients and the tumorectomy for the site of metastasis with irradiation was performed in 2 of the 3 patients, resulting in complete remission. One of 3 patients treated with chemotherapy alone, died. Our satisfactory results suggest the aggressive surgical therapy for the site of metastases in selected patients as indicated is apparently effective with vigorous systemic chemotherapy and radiotherapy.

Although we encounter endometriosis not infrequently, the exact nature of this entity has not yet been determined. In order to study the morphogenesis of this disease, we chose the adenomyotic glandular epithelium as distinct ectopic glands and examined them in the electronmicroscope, comparing them with the proliferative phase of the endometrium and the uterine body adenocarcinoma. Five cases were selected from 76 histologically proven cases of adenomyosis at our University. Epon blocks were ultrasectioned with a Porter MT II microtome, stained with uranium acetate and lead, and observed in a JEM 100 C electronmicroscope. The characteristics of adenomyotic glandular epithelium compared to the normal proliferative endometrium were: 1) expanded smooth nuclear membrane with scattered fine chromatin and zero to one irregularly surfaced nucleoli, 2) in cytoplasm, rough ER and free ribosomes are distinct, mitochondria are round to oval in shape with moderate irregularity, and microfibrillar structures and interdigitations are present in moderate amounts. Although there are few similarities to endometrial cancer, these findings would suggest that adenomyosis may be an intermediate between the normal proliferative endometrium and endometrial cancer.

We investigated the usefulness of the measurement of serum CA125 levels for the diagnosis and therapeutic monitoring of endometriosis. An additional study concentrated on the production of CA125. 1. Elevated levels of serum CA125 were noted in 52 of 66 patients with endometriosis in which the positive rate was 78.8% and mean was 119.8 U/ml. The mean value and positive rate of serum CA125 levels in patients with adenomyosis were higher than those in pelvic endometriosis. 2. The correlation between preoperative serum CA125 levels and the extracted tissue weight was statistically significant. The tissue concentration of CA125 of adenomyosis was 1,479.3 +/- 1,087.1 U/g and that of pelvic endometriosis was 309.7 +/- 23.1 U/g wet weight. 3. The serum CA125 levels in patients with adenomyosis fell postoperatively, and all were below 35 U/ml within two weeks. The serum CA125 levels were below 35 U/ml in 13 out of 15 patients (86.7%) with pelvic endometriosis treated with danazol and the change in the serum CA125 levels was closely related to the clinical course. 4. Clinicopathological states with a high level of serum CA125 were observed in patients with normal and ectopic pregnancy, puerperium, ovarian hyperstimulation syndrome (OHSS) and peritonitis. It was concluded that the measurement of serum CA125 levels was useful in the diagnosis and therapeutic monitoring of endometriosis, and CA125 might be produced and/or secreted not only from the endometrium but also from the peritoneum.